Double zero tillage and foliar phosphorus fertilization coupled with microbial inoculants enhance maize productivity and quality in a maize–wheat rotation

Maize is an important industrial crop where yield and quality enhancement both assume greater importance. Clean production technologies like conservation agriculture and integrated nutrient management hold the key to enhance productivity and quality besides improving soil health and environment. Hence, maize productivity and quality were assessed under a maize–wheat cropping system (MWCS) using four crop-establishment and tillage management practices [FBCT–FBCT (Flat bed–conventional tillage both in maize and wheat); RBCT–RBZT (Raised bed–CT in maize and raised bed–zero tillage in wheat); FBZT–FBZT (FBZT both in maize and wheat); PRBZT–PRBZT (Permanent raised bed–ZT both in maize and wheat], and five P-fertilization practices [P100 (100% soil applied-P); P50 + 2FSP (50% soil applied-P + 2 foliar-sprays of P through 2% DAP both in maize and wheat); P50 + PSB + AM-fungi; P50 + PSB + AMF + 2FSP; and P0 (100% NK with no-P)] in split-plot design replicated-thrice. Double zero-tilled PRBZT–PRBZT system significantly enhanced the maize grain, starch, protein and oil yield by 13.1–19% over conventional FBCT–FBCT. P50 + PSB + AMF + 2FSP, integrating soil applied-P, microbial-inoculants and foliar-P, had significantly higher grain, starch, protein and oil yield by 12.5–17.2% over P100 besides saving 34.7% fertilizer-P both in maize and on cropping-system basis. P50 + PSB + AMF + 2FSP again had significantly higher starch, lysine and tryptophan content by 4.6–10.4% over P100 due to sustained and synchronized P-bioavailability. Higher amylose content (24.1%) was observed in grains under P50 + PSB + AMF + 2FSP, a beneficial trait due to its lower glycemic-index highly required for diabetic patients, where current COVID-19 pandemic further necessitated the use of such dietary ingredients. Double zero-tilled PRBZT–PRBZT reported greater MUFA (oleic acid, 37.1%), MUFA: PUFA ratio and P/S index with 6.9% higher P/S index in corn-oil (an oil quality parameter highly required for heart-health) over RBCT-RBCT. MUFA, MUFA: PUFA ratio and P/S index were also higher under P50 + PSB + AMF + 2FSP; avowing the obvious role of foliar-P and microbial-inoculants in influencing maize fatty acid composition. Overall, double zero-tilled PRBZT–PRBZT with crop residue retention at 6 t/ha per year along with P50 + PSB + AMF + 2FSP while saving 34.7% fertilizer-P in MWCS, may prove beneficial in enhancing maize productivity and quality so as to reinforce the food and nutritional security besides boosting food, corn-oil and starch industry in south-Asia and collateral arid agro-ecologies across the globe.

Under the aegis of United Nations Sustainable Development Goals (SDGs), there is an urgent need to focus both on food and nutritional quality enhancement for eradication of all types of hunger and malnutrition by 2030 especially in under-developed countries 1 . We already know that rice-wheat cropping system (RWCS), a major system in south-Asia in general and India in particular, is a major contributor to the food and nutritional security of the region 2,3 . However, intensive agriculture practices under RWCS especially in the Indo-Gangetic Plains Region (IGPR) coupled with intensive conventional tillage 4,5 , sole use of chemical fertilizers [6][7][8] , over-exploitation of groundwater 9 , and in situ crop residue burning 10,11 ; has led to stagnation in productivity with impaired quality, sub-soil compaction, soil health deterioration, groundwater depletion and gradual degradation of natural resource-base 11,12 . The escalating labour, capital, and energy requirements 4 coupled with receding groundwater table (~ 0.30-0.40 m year −1 ) 13 , erratic rainfall pattern and intermittent droughts 5 , has further triggered the chronic fatigue in RWCS in south-Asian IGPR for over last three decades 5,11 . Rice and wheat crops' residue burning has also long been a major cause of air pollution releasing huge gaseous emission in northern India 10,14 , impairing soil and human health and environment 15 . To deter these ill-effects, crop diversification and conservation agriculture (CA) are two viable options 4,16 , over the policy backed conventional RWCS 17 . Bringing National Policy for Management of Crop Residues 18 in India is again a timely effort which stresses upon in-situ residue management through CA and other sustainable residue management methods 19 . Hence, research priorities integrating clean
Lysine and tryptophan content. The CETMs did not have any significant effect on the lysine content in maize grains, however, the raised-bed CETMs viz. PRBZT-PRBZT and RBCT-RBZT produced higher lysine  Table 2). The PRBZT-PRBZT observed ~ 1.2% higher lysine content over the FBCT-FBCT. The P 50 + PSB + AMF + 2FSP reported significantly higher lysine content (2.74 g kg −1 dry matter) which was followed by P 100 , P 50 + 2FSP, P 50 + PSB + AMF and P 0 , respectively ( Table 2). Integration of P 50 + PSB + AMF + 2FSP exhibited ~ 6.6% higher lysine content over 100% soil applied-P. The CETMs again didn't show any significant influence on tryptophan content, although, double zero-tilled PRBZT-PRBZT and FBZT-FBZT treatments reported comparatively higher tryptophan content over the conventional-tilled FBCT-FBCT system. Among PFPs, P 50 + PSB + AMF + 2FSP had significantly higher tryptophan content by ~ 10.4% over P 100 . In general, tryptophan followed similar trend as that of protein content both for CETMs and PFPs in current study (Table 2).

Discussion
Diversifying the existing dominant RWCS towards viable alternative maize-based systems particularly the MWCS 16,21,35 , the conservation agriculture based CETMs (PRBZT-PRBZT; FBZT-FBZT) along with the appropriate P-fertilization practices (PFPs), could enhance and stabilize the yields besides improving soil health in long-run 17,63 , and more importantly the quality parameters of this potential food and industrial crop of south-Asia. The tillage and input-intensive RWCS in the IGPR of south-Asia is facing multiple production-and resource-vulnerabilities viz. exaggerating decline in crop productivity, groundwater table, input-use efficiencies and soil-health 5,11,17,21 . Henceforth, CA based MWCS has ample potential to combat these assailabilities besides resolving twin challenges of maize productivity and quality enhancement for ushering in food and nutritional security vis-à-vis augmenting industrial applications of this crop in south-Asia. In this study, the CA-based double zero-tilled permanent raised-bed system (PRBZT-PRBZT) with crop residue retention at 6 t ha −1 per year in MWCS had significantly (p < 0.05) higher maize grain yield by 13.1% over the CT-based FBZT-FBZT, and by 5.7-6.4% over the double zero-tilled flat-bed system (FBZT-FBZT) and the single crop basis zero-tilled system in preceding wheat (RBCT-RBZT) across the years (Fig. 1). It could be associated with the positive impact of crop residue retention and zero-tillage on modulation of soil temperature 64 17,27,30 , resulting in better plant growth and yield 11 . Double ZT system provides better soil physical conditions due to less machine trafficking 65 , better seed germination and optimal seedling establishment due to avoidance of hard crust formation on soil surface, a characteristic feature of alluvial soils of IGPR 70 . Higher maize yield under PRB/RB plots (PRBZT-PRBZT, RBCT-RBZT) over the flat-bed CT and ZT plots may also be attributed to better root aeration and root anchorage in raised-beds 7,71 , least water stagnation during rains 27 , and better moisture conservation in rainless spans 72 . Crop residue retention and its slow decomposition enhance the SOC 6 and soil moisture content 27 , both of which are ideal for favorable soil biological activities in ZT 20,73,74 , which eventually augment the nutrient bio-availability 10,75 favoring growth and productivity 67,70,76 . Since, P-fertilization directly influences the root growth and development which in turn improved the vegetative and reproductive growth vis-à-vis maize yield 57,77 . Integration of P 50 + PSB + AMF + 2FSP, a combination of soil applied-P, microbial inoculants (PSB, AMF) and the two foliar-P sprays (2% DAP), had significantly (p < 0.05) higher grain yield by 11.3-17.5% over the soil applied-P 100 and P 0 (Fig. 1). Furthermore, the integrated use of P 50 + PSB + AMF + 2FSP saved ~ 34.7% fertilizer-P over the soil applied-P 100 both in maize alone and on cropping system basis in MWCS. In alkaline soils of semi-arid IGPR, soil applied-P reacts with calcium (Ca) and magnesium (Mg) ions to form Ca and Mg phosphates making P unavailable to plants 57 . Hence, 2 foliar-P sprays at knee-high and pre-tasseling stage of maize proved beneficial for P-absorption through foliage which enhanced the plant growth and photosynthetic activity leading to improved maize yield 58 . Foliar-P skip the P-fixation and leads to higher PUE 62 , which otherwise is an unavoidable fate of soil applied-P in alkaline and acidic soils 61 . Inoculation of maize grains with PSB and AMF along with 50% soil applied-P proved effective even over 100% soil applied-P due to improved P-availability and uptake owing to their synergistic effect on P-solubilization and mobilization of fixed native-and applied-P 51,78 . Furthermore, the AMF mycelia growth greatly enhances the root exploratory area (10-1000 folds), thus, helping in better nutrient and water acquisition 61,79 . Exudation of organic acids/chelating agents by AMF mineralizes the organic residues and manures to release inorganic nutrients with better phyto-availability 65,80 besides enriching soil microbial diversity 7,8 , thus adding to better yields.  (Table 1). This may be attributed to enhanced macro-and micronutrient availability especially P and K 30 , owing to residue decomposition with better nutrient-recycling especially K 10,83 , and mineralization and solubilization of native-and applied-P by the organic acids released from decomposing residues under ZT 28,84 . Likewise, maize under raised-beds (PRBZT-PRBZT, RBCT-RBZT) exhibited higher starch content (~ 66.2%) compared to ~ 64.9% under flat-beds (FBZT-FBZT, FBCT-FBCT) irrespective of tillage practices owing to better root aeration and anchorage for nutrients 71 especially limiting nutrients like P 61 . Thus, ZT based CETMs enhanced the starch content over the conventional-tillage. Among PFPs, significantly (p < 0.05) higher starch (68.9%) was obtained by integrated use of P 50 + PSB + AMF + 2FSP followed by P 50 + 2FSP and least under P 0 . Here, foliar-P fertilization proved beneficial in higher P absorption by maize foliage and its assimilation which enhanced the starch content. Under P-deficiency, starch content decreases because of reduced ATP production in chloroplast resulting in reduced activity of ADPG enzyme, a key enzyme in starch metabolism; so the starch produced in chloroplast was unable to diffuse to cytoplasm as tri-phosphate, thus, resulting in reduced translocation of carbohydrates to grains 82 . Starch content showed positive correlation with grain yield both for CETMs and PFPs, owing to greater role of starch in grain biomass accumulation being influenced by both CETMs 30 , and P-fertilization 38 . The CETM practices again did not show any significant effect on amylose and amylopectin content in maize grains like starch content. Starch biosynthesis is mainly dependant on proteins present in the starch granules 82 , particularly the granule-bound starch synthase I protein (GBSSI) which is involved in amylose synthesis 85 . Hence, enhanced N-availability and protein content in ZT based CETMs might have enhanced the amylose content to some extend over CT based CETMs. Henceforth, higher amylose content under PRBZT-PRBZT and P 50 + PSB + AMF + 2FSP may be associated to improved nutrient availability and acquisition [86][87][88] . Higher amylose content under PRBZT-PRBZT and 50% P + PSB + AMF + 2FSP, considered as a beneficial trait due to its lower glycemic-index, required by diabetic patients 45    www.nature.com/scientificreports/ The ZT based CETMs had higher protein content over the conventional-tillage due to residue retention (3-6 t ha −1 per year) which on decomposition and mineralization enhanced the N-availability and uptake to synthesize amino acids and proteins 20,89,90 . Higher protein content in maize may also be associated with preferential deposition of zein protein over other endosperm proteins 89,91 . Protein yield was higher under ZT based CETMs compared to CT plots due to improved nutrient availability and soil health 92,93 , optimal soil moisture status and better root activities 88,94 . The PFPs had significant influence on protein content and protein yield owing to the vital role of P in protein biosynthesis and energy relations 81,82 . Furthermore, the P and N are found to have synergistic effect, thus, integrated use of P 50 + PSB + AMF + 2FSP might have significantly enhanced the N uptake and assimilation 95 , leading to greater protein content and protein yield 96 . Nutritionally essential amino acids viz. lysine and tryptophan are highly important to improve maize grain quality 97 . Here, different CETMs had non-significant effect on lysine and tryptophan content. It may be strengthened with the fact that an increase in grain-N content as a result of improved N-availability is accompanied by decrease in the relative lysine content of grain proteins 98 . On the other hand, P indirectly influences the lysine content because when P-supply is reduced, it results in reduced grain yield but with increased grain-N concentration; thus, leads to reduced lysine content in grains 98 . Although under optimal P-fertilization, here P 50 + PSB + AMF + 2FSP, the grain yield increases which results in reduced grain-N content due to dilution effect, which in turn, increases the lysine content under optimal or excess P-supply 99 . Henceforth, a similar pattern was observed for lysine content under PFPs in current study. Since, P and Zn are found to have antagonistic effects, so the P plays a vital role in tryptophan production 100 . The Zn is involved in various oxidation-reduction reactions 101 ; thereby, Zn-deficiency leads to oxidation of auxins and reduction of tryptophan 102 . As, tryptophan is the precursor of auxins 97 , hence, tryptophan was higher under P 50 + PSB + AMF + 2FSP over soil applied-P 100 ; because of reduced Zn-uptake under soil applied-P 100 compared to P 50 + PSB + AMF + 2FSP, a combination of soil, microbial and foliar-P application which had an advantage over soil applied-P owing to reduced competition for Zn-uptake 100,103 . Positive correlation of protein, lysine and tryptophan content with the grain yield both under CETMs and PFPs, further emphasize the importance of ZT based CETMs and integrated use of P 50 + PSB + AMF + 2FSP in enhancement of protein, lysine and tryptophan content as well as protein yield. Thus, deployment of such CPTs may altogether boost farm productivity and quality for better profitability of resource-poor south-Asian farmers and the maize based food, feed, and pharmaceutical industry in the region.
Different CETM practices had non-significant effect on oil content in maize grains although ZT based CETMs proved superior over conventional-tillage; where PRBZT-PRBZT had higher oil content due to crop residues decomposition 104 , which slowly released the essential nutrients (soluble-P and S-compounds) into rhizosphere which later became available to plants specifically during reproductive phase 105 . Sulfur (S) is a key element in chlorophyll formation, yield enhancement and oil synthesis 57 . Furthermore, S-concentration and S-uptake has a strong synergistic relationship with P in plants 57,106 . Positive correlation and heatmap clustering between oil content and grain P uptake under PFPs has further strengthens this fact. As per an estimate, the wheat straw of 2700 kg ha −1 , on average add ~ 28 kg N, 4.5 kg P, 52 kg K and 6 kg S ha −1 under ZT system; on the other hand, this advantage may lack in CT system 107 . Hence, crop residues decomposition released both P and S while additional application of foliar-P augmented S uptake from the soil, thereby, enhancing the oil content. The P 50 + PSB + AMF + 2FSP exhibited significantly higher oil content and oil yield over other PFPs which may be accrued to the fact that P directly participates in synthesis of oils, fats and phospholipids 108 , besides its vital role in S acquisition 57 . Better plant nutrition under CA based CETMs and integrated P-fertilization practices though caused a slight improvement in grain oil content in current study 109 , but harnessed greater oil production per unit area because of enhanced grain yield 38 . Positive correlation between oil content and maize grain yield has strongly established this relationship in current study. Hence, maize cultivation under PRBZT-PRBZT system along with P 50 + PSB + AMF + 2FSP may lead to higher corn-oil productivity which may cut down the oil imports by the developing nations like India.
The ZT and CT based CETMs exhibited non-significant effect on the composition of fatty acids viz. saturated fatty acids (SFA), mono unsaturated fatty acids (MUFA) and poly unsaturated fatty acid (PUFA) like oil content. However, these fatty acids were greatly influenced by PFPs both under CT and ZT systems with pattern of fatty acid composition as PUFA > MUFA > SFA both under CETMs and PFPs 110 . With increase in P-supply, SFA (Palmitic acid + Stearic acid) content decreased while MUFA (Oleic acid) content increased considerably, again as an indicator of better oil quality 42 . These observations are in agreement with the findings of Ray 38 , who observed similar findings with respect to SFA and MUFA content while using higher doses of plant nutrients. The PUFA (linoleic acid) content were higher under P 0 in current study; which further corroborate with the findings of Krueger 56 , who observed an increase in linoleic acid with the P-omission. In current study, the influence of different CETMs and PFPs on the status of individual fatty acid may not lead to definite conclusion about the overall fatty acid composition in corn-oil. Thus, various fatty acid ratios were estimated to draw logical conclusions. Among CETMs, double zero-tilled PRBZT-PRBZT had significantly higher MUFA: PUFA ratio and P/S index over CT plots which show better oil quality under ZT system owing to better N-supply encouraging carbon chain elongation in linoleic acid (PUFA) and oleic acid (MUFA) [111][112][113] . The P/S index is a vital factor among all parameters as it represents the nutritional value of edible oils 38 . Here, P/S index was found to be > 1.0 irrespective of CETMs and PFPs, which sufficiently indicated the better nutritional value of corn-oil with reduced tendency of deposition of lipids in the human body 114 . It was found that ZT based CETMs and P 50 + PSB + AMF + 2FSP exhibited higher P/S index over the CT system and P 0 . It is reported that with an increase in unsaturation content and a degree in fatty acid, the susceptibility to oil oxidation increases; thus, releasing free radicals causing offflavor and reduced nutritional quality 38 . On average, oleic acid is 25-times less vulnerable to oxidation compared to linoleic acid, while linoleic acid is 2-times less susceptible compared to linolenic acid because of an increase in bond association energy as compared to linolenic acid 115 . As, ZT based CETMs and P 50 + PSB + AMF + 2FSP had higher oleic acid and lowest linoleic acid content, a positive sign for producing good quality corn-oil having Scientific Reports | (2022) 12:3161 | https://doi.org/10.1038/s41598-022-07148-w www.nature.com/scientificreports/ less susceptibility to oxidation which may help in flourishing the corn-oil industry. Linolenic acid is susceptible to oxidation and causes adverse effect on human health like cardiovascular diseases and improper brain development 116,117 . As corn-oil contained negligible amount (< 1%) of linolenic acid in current study, hence, it would not exert any adverse effect on human health 38 . Under P 0 , higher oleic desaturation ratio (ODR) and lesser MUFA: PUFA ratio compared to other PFPs again point out a better quality corn-oil 105 . Higher ODR indicates better and longer shelf-life of corn-oil; while lower ODR inhibits the subsequent desaturation steps which lead to reduced linolenic acid content 112 . The P-fertilization considerably increased the ODR and MUFA: PUFA ratio under P 50 + 2FSP, P 50 + PSB + AMF and sole P 100 ; but P 50 + PSB + AMF + 2FSP showed slight reduction in ODR and an increase in MUFA: PUFA ratio may be due to enhanced P-availability over P 0 . The MUFA: PUFA ratio is directly linked with the oxidative stability and nutritional properties of the oil 118 , thus, indicating that optimal P-nutrition and the ZT system both may improve the oil quality due to sustained and synchronized P bio-availability throughout crop season. Significantly higher P/S index under P 50 + PSB + AMF + 2FSP is another indicator of better nutritional value of edible maize oil 38 . The heatmap also demonstrated that optimal P-nutrition and ZT system proved highly promising in producing good quality corn-oil, a good indication for corn-oil industry to target health conscious clientele 114 . The CETMs and PFPs showed significant influence on P uptake in maize grains with greater magnitude under PRBZT-PRBZT and P 50 + PSB + AMF + 2FSP, owing to higher grain-P concentrations and maize yield in these treatments. Higher grain-P uptake in PRBZT-PRBZT is attributed to affirmative effects of crop residue retention which added substantial amount of nutrients including P in soil while improving soil physicochemical and microbiological properties compared to CT plots 27,72,119 . The P-fertilization along with PSB and AMF vis-à-vis foliar-P had a significant influence on grain-P uptake as a result of optimal P bio-availability 61 , better root and shoot system 11 , enhanced native and applied-P solubilization and mobilization 65,120 , and foliar-P supplementation 58 ; which collectively led to higher P uptake 121 . Thus, better the P-fertilization better is the P uptake by the crop and its subsequent accumulation in grains 62 . The P-fertilization in adequate amounts is essential for root and shoot development, seed formation and biochemical reactions viz., synthesis of proteins, oils and fats, phospholipids and energy relations, thus, it played a vital role in enhancing the maize quality 108 . That's why, the quality parameters of maize viz., starch, protein, lysine, tryptophan, MUFA, and MUFA: PUFA ratio had positive correlation with grain-P uptake. Contrary to that, the amylopectin, PUFA, SFA, ODR and SFA: UFA ratio showed an inverse relationship with the grain-P uptake owing to complex interrelationships with their counterpart constituents 82,85,108 , and with varying P supplies as reported by various researchers [86][87][88]99,100 . The heatmap biclustering validated the superiority of CA-based PRBZT-PRBZT and FBZT-FBZT systems in combination with two PFPs viz. P 50 + PSB + AMF + 2FSP and P 50 + PSB + AMF in enhancing the grain, protein and oil yield as well as starch, amylose, lysine and tryptophan content; which demonstrate the sustainability of CA based crop management over the conventional agriculture while integrating P 50 + PSB + AMF + 2FSP in MWCS. It is tangibly evident from the PCA analysis and heatmap biclustering that the M 4 S 4 , a combination of double zero-tilled PRBZT-PRBZT system in combination with P 50 + PSB + AMF + 2FSP may prove highly sustainable for realizing higher maize grain yield and quality under a maize-wheat cropping system in a semi-arid agro-ecology. Thus, clean production technologies like double zero-tilled PRBZT-PRBZT along with P 50 + PSB + AMF + 2FSP not only enhanced the maize yield significantly while saving ~ 34.7% fertilizer-P both in maize and MWCS, but they also augmented the maize quality parameters to reinforce the food and nutritional security besides boosting food, corn-oil and starch industry in the south-Asia.

Conclusions
In order to safeguard the food and nutritional security of millions of south-Asian families concurrently conserving the soil, environment and natural resources, the application of clean production technologies (CPTs) like CA-based CETMs (PRBZT-PRBZT/FBZT-FBZT) that allows rapidly increases of yield and food quality should be a norm, not the exception. In our study, the production technology of the PRBZT-PRBZT/FBZT-FBZT along with integrated use of P 50 + PSB + AMF + 2FSP in MWCS proved to be excelled in the maize yield and quality parameters. On average, double zero-tilled PRBZT-PRBZT system and P 50 + PSB + AMF + 2FSP both significantly enhanced the maize grain, starch, protein and oil yield by 13.1-19% and 12.5-17.2%, over their respective counterpart treatments i.e. FBCT-FBCT and 100% soil applied-P (P 100 ); while concurrently saving ~ 34.7% fertilizer-P both in maize (20.8 kg P 2 O 5 /ha) and on cropping system basis (41.6 kg P 2 O 5 /ha). Integrated use of P 50 + PSB + AMF + 2FSP had significantly higher starch, amylose, protein, lysine, tryptophan and oil content by 1.2-10.4% over the 100% soil applied-P due to sustained and synchronized P bio-availability to the crop. The PRBZT-PRBZT had greater MUFA (oleic acid, 37.1%), MUFA: PUFA ratio (0.79) and P/S index (3.09). The ODR, MUFA: PUFA ratio and P/S index responded positively and significantly to P-fertilization practices. Double zero-tilled PRBZT-PRBZT system concurred with residue retention at 6 t ha −1 per year along with P 50 + PSB + AMF + 2FSP while saving ~ 34.7% fertilizer-P in MWCS, proved as a potential clean production technology for enhancing the maize productivity and quality. Accordingly, deserves strong recommendation to augment maize yield and quality besides augmenting safe industrial uses in maize based industries, climateresilience and farmers' well-being in semi-arid IGPR in south-Asia and similar agro-ecologies across the globe.  10,11). Average annual rainfall is ~ 650 mm 80% of which is received through 'South-West Monsoons' during July-September and the rest during 'Western Disturbances' from December to February. Mean annual evaporation is ~ 850 mm. Physico-chemical analysis of composite soil samples (0-15 cm depth) was done at the start of the experiment using standard procedures (Table 4). Soil had pH 8.0, oxidizable soil organic-C 0.421%, alkaline KMnO 4 oxidizable-N 137.9 kg ha −1 , 0.5 M NaHCO 3 extractable-P 12.9 kg ha −1 and 1 N NH4OAc extractable-K 302.8 kg ha −1 .

Materials and methods
The  Maize grain yield, protein content and protein yield. After harvesting, the maize crop from net-plots was sun-dried, threshed plot-wise, grains cleaned and sun-dried till 10% seed moisture was obtained. Grain yield (t ha −1 ) was estimated using standard procedures 132 . Nitrogen content (%) in maize grains was determined using standard procedure 132 . Protein content (%) in maize grains was calculated by multiplying grain-N content (%) by the factor 6.25 while protein yield (kg ha −1 ) in maize grains was calculated by using following formula: Starch estimation. A grain sample of 0.4 g was homogenized in hot 80% ethanol to remove sugars. The residues retained after centrifugation were washed repeatedly with hot ethanol (80%) till the washing is colorless. The residues were dried and the extraction was done from the dried samples with the application of 5 mL water and 6.5 mL of percholoric acid (52%). The 0° C temperature was maintained for 20 min (min) and then samples were put under centrifugation at 10,000 rpm for 8 min. The supernatant was decanted and kept for starch estimation. The extraction was repeated 2-3 times for full and final extraction. With the addition of distilled water, final volume of the pooled-up supernatant was made to 100 mL. The 0.1 mL of supernatant was pipetted-out and the volume was made-up to 1.0 mL with distilled water. Similarly for reference, different aliquots of standard glucose solution were taken and volume was made-up to 1.0 mL using distilled water. The 4.0 mL of anthrone reagent was added to each tube and heated for 8 min in water bath. Intensity of color, green to dark green, was recorded at 630 nm 133 . The glucose concentration of the samples was determined using the calibration curve and the values obtained were multiplied by a factor 0.9 to quantify the starch content (%). Starch yield (kg ha −1 ) in maize grains was calculated by using following formula: Amylose and amylopectin content. Maize grains from different plots were ground to make fine powder with particle size of 500 µ after milling. 100 mg of powdered samples was added with a mixture of ethanol and 1 M NaOH (1 mL + 10 mL) and was left as such overnight. Subsequently, distilled water was added to sample Protein yield (kg/ha) = Protein content (%) × Grain yield (kg/ha) 100 Starch yield (kg/ha) = Starch content (%) × Grain yield (kg/ha) 100 www.nature.com/scientificreports/ solution to make the final volume to 100 mL. An aliquot of 2.5 mL of extract was mixed with 20 mL distilled water and 3 drops of Phenolphthalein, where by the solution changes into pink-color. On addition of 0.1 M HCl drop by drop, the pink color disappears. To the treated sample, 1 mL of iodine reagent was added and volume was made-up to 50 mL by adding distilled water and then absorbance was recorded at 590 nm with reference to blank (1 mL iodine reagent diluted to 50 mL with distilled water). The amylose content in maize grains was determined using standard curve derived from potato amylose 134 . Standard amylose solution was prepared by dissolving 100 mg in 10 mL of 1 M NaOH and making up to 100 mL final volume. The amount of amylose in samples was determined by using standard curve prepared from amylase (0.2, 0.4, 0.6, 0.8 and 1.0 mL) against a blank for which dilute 1 mL of iodine reagent to 50 mL with water. The relevant calculations were done using following formula: Since, 2.5 mL of the test solution = x mg amylose; therefore, 100 mL contains = x 2.5 × 100 . The amylopectin content (%) in maize grains was determined by subtracting the amylose content from the total starch content 135 . Lysine and tryptophan estimation. The 5 mL papain solution was added to 100 g defatted maize grain sample and incubated at 65 °C overnight. It was cooled down to room temperature, centrifuged and decanted. Carbonate buffer (0.5 mL, pH 9.0) and copper phosphate suspension (0.5 mL) was added to 1 mL digest; after that the mixture was shaken for 5 min in a vortex mix and centrifuged. To 1 mL supernatant 0.1 mL of pyridine reagent was added, mixed well and shaken for 2 h. Then after adding 5 mL of 1.2 M HCl and mixing, extraction was done 3 times with 5 mL ethyl acetate, and ethyl acetate top layer was discarded. The absorbance of aqueous layer was read at 390 nm 136 . The standard lysine solution was prepared by dissolving 62.5 mg lysine mono hydrochloride in 50 mL carbonate buffer. For preparing a standard curve, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the standard lysine solution was pipetted out in different test tubes and final volume of 1 mL was made using carbonate buffer. Later, added 4 mL papain to each tube and mixed thoroughly. Now, 1 mL was pipetted out and 0.5 mL of amino acid mixture and 0.5 mL of copper phosphate suspension were added to it. Afterwards, 1 mL solution from each test tube was transferred to other test tubes and adding 0.5 mL amino acid mixture and 0.5 mL copper phosphate suspension to each one. The above steps were repeated as followed in case of samples and the absorbance of aqueous layer was read at 390 nm 136 . The lysine content in maize samples was determined from standard curve and results were expressed as g kg −1 dry matter.
For estimation of tryptophan, 15 mg defatted maize grain sample was taken in three different 50 mL conical flasks. In 2 flasks, 30 mg of p-dimethyl amino benzaldehyde was added. Third flask acted as the blank. To all the flasks, 9.5 M H 2 SO 4 solution was added. The flasks were kept in dark for 20 h at 30 °C followed by addition of 0.1 mL of 0.045% NaNO 2 solution to each flask. After mixing, the flasks were again kept for 30 min at room temperature. After centrifugation, the absorbance of blue color of the solution was measured at 660 nm 137 . A standard curve of tryptophan was prepared by taking various concentrations (10 to 60 µg mL) of standard tryptophan solution; the volume was made up to 0.6 mL by adding distilled water followed by addition of 9.4 mL of 9.5 M H 2 SO 4 solution slowly and mixed gently. Same steps were followed for the standard solutions. Tryptophan content in the samples was determined from standard curve and expressed as µg g −1 .
Oil content and oil yield. Oil content (%) in maize grains was determined by petroleum ether extraction in a Soxhlet apparatus for 16 h according to AOAC procedure 948.22 138 . Oil yield (kg ha −1 ) in maize grains was calculated by using following formula: Fatty acid analysis and fatty acid ratios. The 100 mg powdered maize grain samples were defatted with solvent mixture of Chloroform:Hexane:Methanol (8:5:2 v/v) for fatty acid analysis. The extracts were dried under a stream of nitrogen and fatty acids were converted into methyl-esters using 0.5 M KOH and 0.5 M HCl. Fatty acids were separated using Gas Chromatography-Mass Spectrometry (GC-MS) following the method as suggested by Kumar and Dhillon 139 . Separation of fatty acids viz. Palmitic acid, Stearic acid, Oleic acid and Linoleic acid was carried-out using HP Innowax capillary column (30 m × 0.32 m × 0.5 µm). The separated peaks were identified on the basis of retention time of standard fatty acid peaks and confirmed using GC-MS library. Besides fatty acid synthesis, different fatty acid ratios viz. ODR, MUFA: PUFA, SFA: UFA and PUFA: SFA were also worked-out using standard formulae 38 . These ratios were calculated excluding the linolenic acid because its contribution to total fatty acid composition was < 1% in maize grain oil. Phosphorus content and its uptake in maize grains. Concentration of P in maize grains was determined by using the Vanadomolybdo-phosphoric acid yellow colour method at 420 nm wavelength on a UV-VIS spectrophotometer. From P content (%) in plants, P uptake (kg ha −1 ) was computed using the formula given below: Statistical analysis. The data related to each parameter were analyzed as per the procedure of analysis of variance (ANOVA) to determine treatment effects through Tukey's honestly significant difference test as a post hoc mean separation test (p < 0.05) by using SAS 9.1 software (SAS Institute, Cary, NC). Tukey's procedure was used where ANOVA was found significant (Supplementary Tables S1 and S2). A two-dimensional heatmap with hierarchical clustering of treatment-by-traits was drawn using R-software package 'gplots' developed by Warnes et al. 141 . To reduce the complexity of relationship, a data reduction technique was performed using principal component analysis (PCA) implemented in the R package 'Factoextra' and 'FactoMineR' , and thereby resulting PC scores were plotted 142